Fluoride's link to bone and joint cancers

by Calabrese E, PhD., Evaluation of the National Toxicology Program (NTP) Cancer Bioassay on Sodium Fluoride, 1991 Jun (Amherst), commissioned by the East Bay Municipal Utility District (provides water for Oakland and area)

INTRODUCTION
Early in 1990 it was reported in the national press that a soon to be released cancer bioassay conducted by the U.S. NTP [National Toxicology Report] would indicated that NaF was a carcinogen in rats. This created considerable debate in the scientific, public health, regulatory and dental communities. Of great concern was that the debate preceded the release of the NTP report, thereby creating even greater anxiety since exposure to fluoride is so widespread in the U.S. and serves as the cornerstone for USPHS programs for dental caries prevention via community drinking water fluoridation programs as well as via many commercial products such as toothpastes and mouth washes.

As a result of the subsequent release of the NTP report on the fluoride cancer bioassay, both government and private sector organizations independently set forth to assess not only the NTP bioassay but all other relevant toxicological and epidemiological studies on NaF. These studies were then set within the context of the societal benefits of fluoridation treatment programs. The cancer bioassay of fluoride, the broader scientific literature on fluoride as well as that of the ad hoc PHS panel that was convened because of the cancer concern generated by unofficial reports that NaF was a carcinogen.

OVERVIEW OF NTP BIOASSAY PROCEDURE
In assessing the NTP NaF cancer bioassay it is essential that one understand what such bioassays are, what their goals are, how they should be interpreted and what relevance they have to humans. Once these four points are understood, it will be possible to then place the NaF bioassay data in proper context.

A. What is an NTP Bioassay
An NTP bioassay is a study conducted under the auspices of the US NTP to ascertain the potential of selected agents to cause cancer in an animal model. Typically the studies are conducted for a two-year period with a specific strain of mice and rats. These two species of rodents were selected principally because of practical considerations including their relatively short life-span, small size, susceptibility to developing tumors, and experience concerning their care, maintenance, and their susceptibility to infectious disease. The specific strains of animals most frequently used by the NTP are the B63F1 mouse and the Fisher (F) 344 rat. The reasons for animal model selection are dominated, therefore, by practical considerations with much less consideration given to their potential for both qualitative and quantitative extrapolation to humans.

The duration of the NTP bioassay for mice and rats is typically 104 weeks. Two important aspects need to be understood within the context of the two-year study duration. The rodents can live considerably longer than two years if properly maintained. However, if the studies were prolonged it would result in the control group, that is the unexposed comparison, acquiring a higher disease (cancer) incidence and this would make it harder to uncover treatment effects in the exposed individuals unless sample size were markedly increased.

A second aspect that needs to be considered is that the exposure only covers from the time immediately after weaning (8 weeks of age to 2 years later). Mice and rats in embryonic, fetal and neonatal stages are not exposed in NTP bioassays. The reason this is potentially important is that the developing organism is believed to be at appreciably greater risk to developing cancer from genotoxic agents than adults. [as are children] Very young animals aren't used in NTP bioassays in part due to the difficulty in developing appropriate dosages since susceptibility to acutely toxic effects of these agents could markedly change every few days. It is also extremely difficult to expose neonatal animals to agents given their very small size and dependence on feeding by the mother. This lack of exposure during the critical development stages is an important limitation of the NTP bioassay which must be taken into consideration when interpreting the findings of such studies.

B. The Goal of the NTP Bioassay
The NTP bioassay is fundamentally a hypothesis testing program which sets forth to answer the following question: does the agent cause an enhanced incidence of cancer at any site under the conditions of the study. the study design incorporates factors that allow this goal to be assessed such as employing the use of multiple doses to determine possible dose-response relationships. this is a potentially powerful feature to the bioassay since dose-related trends can be defined across a broad spectrum of exposures. Only three dosage levels are usually selected based on practical considerations. Consequently, genuinely insightful dose-response relationships are often not derived from NTP bioassays. This is often further compromised by the inappropriate selection of the highest dose [i.e. maximum tolerated dose (MTD)], which is the highest exposure level the animal can sustain for the 104 week period without sustaining a significant decrease in body weight (usually about 10%), reduction in life-span, or other important preliminary indications of toxicity. It is not uncommon that investigators exceed the MTD in cancer bioassays. When this occurs the MTD usually isn't very helpful in contributing to defining the dose-response relationship. More importantly, eliminating the MTD group from the study because of unacceptable toxicity reduces the number of effective dosages from three to two and greatly compromises the possibility to discern dose-related trends.

The NTP bioassays place heavy reliance on statistical analysis of the data. In order to achieve adequate statistical power the bioassay utilizes 50 animals per sex per dosage level represents a practical compromise between how large a sample size is needed for statistical analysis in relationship to how much the study costs to conduct. this type of study design with a limited sample size cannot effectively discern cancers of normally low incidence. For example, if a tumor incidence were to occur in one out of 250 animals it would not be detectable in this study. Yet such an incidence in the U.S. population would be of considerable public health concern. The NTP bioassay tries to overcome this limitation by "pushing" the dose to the highest level that animals can sustain (i.e. MTD) for the two-year period. The NTP bioassay includes comprehensive pathological analyses of all tissues and has the tissues assessed in a "blind" fashion so that results will not be biased. All things considered, one can expect the NTP bioassay findings to be of very high quality, and have undergone an extremely vigourous peer-review. ...

[editor's note: and this is wherein the controversy lies. The original laboratory which conducted the study under the auspices of the NTP was Battelle. They found significant dose-related occurrences of bone cancers and oral tumors (more to follow on this). It is the outside contractors which downgraded almost all of Battelle's results. See William Marcus Memo]

[Excerpts:] CANCER BIOASSAYBone Cancer (Osteosarcoma)

... The NTP concluded that the male rat data on osteosarcoma revealed that a "weak association may exist between the occurrence of these neoplasms and the administration of NaF". The NTP concluded that there was equivocal evidence of a carcinogenic activity, meaning that the studies displayed a marginal increase of neoplasms that my abe related to chemical administration.

In considering the conclusions of the NTP on this issue one can initially take issue with the terminology employed. For example, equivocal is a word that should not be applied to findings where statistically significant trend relationships exist and where the response has substantial biologically plausibility. The fact that fluoride is a potential mutagen/clastogen, concentrates in the bone, and is known to stimulate bone development (Erikson et al., 1988), suggests that bone alterations need to be carefully considered. The linkage of the site of deposition and biological activity with bone cancer outcome all speak to the plausibility issue. This reasoning challenges the NTP conclusion to place the evidence on NaF in a category reserved for uncertain findings.
ORAL CANCER

The other cancerous incidence data of relevance in the rat were the findings that combined oral papilloma and carcinoma in the male (3/80 vs. 0/80 in controls) and female rats (3/80 vs 1/80 in controls) were marginally increased at the 175 ppm group. The increase was not statistically significant in any pair wise comparisons but approached statistical significance in males in the trend test (P=0.09). Squamous cell neoplasms of the oral mucosa occur at a low incidence of 0.7% in males and 0.8% in females in historical controls. The 0/80 incidence in males controls and 1 of 80 in female controls indicates that the controls acted in an expected fashion. The NTP did not consider these tumors chemically related because 1) a squamous cell carcinoma was seen in one pair control group and one control group female, 2) the increased incidence were not statistically significant, 3) the highest historical control incidence was 4%, 4) there was no increased focal hyperplasia in the oral mucosa. The argument to dismiss these findings by NTP has merit but is not convincing since both males and females showed a dose related presence of a squamous cell carcinoma in a comparison group (1/144) can not be used to dismiss the possibility that the 3/80 historical control value of 4% is of questionable relevance since the historical control groups had elevated NaF in their diets and controls. The present judgement concerning oral tumors, using the NTP terminology, is more closely associated with an equivocal finding, that is where marginal increases were observed that might be treatment related. This conclusion is more consistent with a judgement that the relationship is possible but not yet probable. ...[emphasis added]

[editor's note: oral cancer rates in humans have sky-rocketed in the last few decades, despite the fact that smoking is on the decrease. The proliferation of topical fluorides and fluoride intake has vastly increased Coincidence?]

CONCLUSION

The great concern to the public was that the report of the NaF NTP bioassay indicates that male rats developed bone cancer in response to treatment. however, after the commotion settled somewhat and the final report released, the conclusions of the NTP expert committee indicated that there was no clear evidence that NaF caused cancer in mice and rats. The major concern about bone cancer in male rats was "down-graded" to that of equivocal (uncertain findings where the expert committee would not support a conclusion that the observed increase in bone cancer was conclusively related to chemical treatment.

The present analysis question this conclusion on several grounds. First, the terminology of the NTP to classify evidence for carcinogenicity is at best unsatisfactory and confusing when it comes to the equivocal category. The term equivocal is generally understood to mean, uncertain, with an equal amount of evidence on both sides of the matter, an unresolved issue. Equivocal in NTP bioassay language are studies showing a marginal increase of neoplasms that may be chemically related. A study which displays a statistically significant increase in malignant bone tumors by a chemical that is active in and concentrates in bones is simply not consistent with what most people would call equivocal. The fact that fluoride has genotoxic potential in human cells is consistent with the cancer findings without restricting its tumorigenic potential to solely genotoxic mechanisms. While the lack of response in female rats and mice in the NTP assay may be used as an argument to restrict the weight of evidence for NaF to be judged as a bone carcinogen, the only previous chemical (i.e. acromycin) determine by NTP to be a bone carcinogen (osteosarcoma) likewise displayed its response in male but not female rats (NTP, 19789). Furthermore, it should be noted that osteosarcomas in humans occur with a higher frequency in males than females. thus, the lack of a response in female rats cannot be used to conclude that such a response in males could not happen nor that it might even be unlikely. The female rat may simply not be a good predictor of susceptibility to osteosarcomas for male rats for acromycin and NaF.

Of particular concern and in great need of emphasis is that the bone fluoride levels observed in the NTP bioassay at the high dose group were similar to human bone samples taken from people who had lived for at least 10 years in an area with an average fluoride level of 4 ppm in drinking water (Zipkin et al., 1958), the U.S. EPA national primary drinking water standard for fluoride. This information is striking since most NTP bioassays would be expected to use dosage levels that grossly exceed expected human exposures. For example, exposure top TCE in the NTP bioassay exceeded typical human exposures by about 100,000-fold!

These collective findings indicate that the decision by the NTP to classify the male F344 rat osteosarcoma findings as equivocal is inappropriate. It should be noted that a special task force (i.e., ad hoc committee on fluoride) was convened by the assistant secretary for health, and human services Dr. James O. Mason, to review both the benefits and risks of water fluoridation and other sources of fluoride. This task force was comprised of highly prestigious representatives from all PHS agencies with responsibilities for environmental health programs in research, in technical assistance to state and local health agencies, in health care or in regulation. [editor's note: however, many of these people were well-known committed fluoridation proponents] The ad hoc Committee agreed with the interpretations of the NTP and concluded that the available valid animal cancer studies (i.e., NTP and Proctor and Gamble, Inc.) failed to establish an association between NaF and cancer. They supported this conclusion by noting that only the male rat in the NTP study (1 of 8 individual sex/species groups available for assessment) showed any evidence of significant malignant tumor formation and that the only positive study (male rates) was judged by the NTP and its external peer-review committee as only equivocal. Thus, on a weight of evidence basis no link was found.

[editor's note: the PHS review committee did not view the original NTP data, they reviewed the downgraded NTP results. more on this to follow]

Proctor & Gamble, Inc. reported the findings of two cancer bioassays that had significant differences as compared to the NTP bioassays. More specifically, Sprague-Dawley rats and CD- 1 mice were used by Proctor & Gamble while F344 rats and B6C3F1 mice were used by NTP. The NTP administered the NaF via drinking water while the Proctor & Gamble study administered the NaF in the diet. The diets used in the two studies also had significant differences. The Proctor & Gamble diet was a semi-synthetic low fluoride diet which was believed to have nutrition inadequacy based on an abnormal appearance of the liver and gastrointestinal tract. The Proctor & Gamble studies also were conducted over higher dosage range.

In contrast to the generally negative NTP mouse bioassay, the Proctor & Gamble mouse study revealed a highly significant increase in osteomas, a benign tumor of the bone, in both males and females. No malignant bone cancers were reported. The occurrence of the osteomas appeared to be confounded by the presence of Type C retrovirus in both control and treatment groups. however, it was not clear whether the presence of the virus played a role in the development of the osteomas. The Sprague-Dawley rat study did not show statistically significant treatment related effects. However, it should be noted that 0/140 combined male and female control rats developed osteosarcoma while a total 3/420 combined treated male and female rats has osteosarcomas.

These results question the conclusion of the Ad Hoc group that the two animal bioassays (i.e. NTP and P&G) fail to establish an association between NaF and cancer. The ad hoc committee agreed that only 1 of 8 comparisons showed an association and that one (i.e., male rat NTP bioassay) was only an equivocal relationship. [emphasis added]

The present interpretation not only challenges the conclusion that the bone cancer in the F344 was equivocal but that it is inappropriate to dismiss the highly significant relationship of NaF exposure to osteoma formation in mice as not being related to a cancerous lesion. This is a benign lesion occurring in both sexes that is treatment related. It is a finding potentially confounded by a viral contamination and this nonmalignant lesion is of uncertain relationship to osteosarcoma development. Nonetheless, the ad hoc committee was unjustified in structuring the debate to only include malignant tumor formation. The case could certainly be made that at least three of the eight comparisons showed significant evidence of tumor formation in a site that both concentrated the agent and where the agent is biologically active. Thus, the ad hoc committee was too restrictive in defining the debate only in terms of malignancy and not tumorigenicity. This is not a proper position for a PHS committee.

[editor's note: this independent evaluation was completed before the epidemiological data from New Jersey found a statistically significant association between fluoridation and osteosarcoma rates in young males]

Note by Elke: The SEER data by Hoover et al., showed a significant association between osteosarcoma rates and fluoridation. This finding was discounted by the USPHS on the basis that there was an absence of a linear trend. In other words, higher cancer rates would be expected the longer the water was fluoridated. However, given the fact that rapidly growing bones in young males are most susceptible to the development of osteosarcoma, and that fluoride is a known toxin to bones, a potent enzyme inhibitor, and may act as a cancer promoter rather than an initiator, the SEER data is much more significant than first thought. If fluoride acts as a promoter (NJ study -- Cohn PD), the duration/latency assumption is not warranted. The low-level, long term characteristics of fluoride exposure are consistent with the conditions for tumour promotion seen in other chemicals (see also Chinese study, and Jones CA et al.,)